Conditioned reflex analysis of functional connections between the hippocampus and limbic system structures

Instrumental defensive conditioned reflex elaborated in dogs to stimulation of the dorsal and ventral hippocampus, also appeared, due to generalization, in response to stimulation of a number of limbic structures. Two types of changes in the generalization effects (estimated by parameters of motor conditioned reaction) were observed in the course of conditioned reflex stabilization: enhancement (in response to stimulation of the lateral hypothalamus, lateral nucleus of septum, limbic cortex) and weakening toward complete disappearance (in response to stimulation of the medial nucleus of amygdala and medial hypothalamus). Manifestation of the generalization phenomenon from the brain structures, which are not involved initially into conditioned activity, suggests the existence of close functional connections between these structures and the hippocampus.     

Neurophysiologic analysis of the critical period of delay of a reinforcing stimulus in the dog

At interval of less than 100 ms between indifferent sound and unconditioned electrocutaneous stimulation, classical defensive conditioned reflex is not elaborated in dogs, in full correspondence with literature data. But intracortical temporary connection is established; this fact is shown by electrographic phenomena, the most specific and demonstrative among them is "conditioned evoked potential". The absence of behavioural (peripheral) and presence of electrographic (central) manifestations allow to assume that for the formation of the conditioned reflex of full value the involvement is necessary of additional activational (motivational) mechanisms, connected with reticular and (or) limbic subcortical structures which do not react to the conditioned stimulus at time deficit in conditions of microdelay.     

Involvement of mesencephalic reticular formation neurons in cat conditioned reflexes

Traditional defensive and operant food reflexes were used to investigate neuronal responses of the mesencephalic reticular formation. It was found that these neurons may be divided into different groups according to function, depending on how they respond to positive conditioning stimuli. Of the two main groups of neurons with sustained tonic reactions one is activated in response to positive acoustic conditioning stimulation; it no longer reacts to the same stimulus after extinction of the reflex, while the other only becomes involved in response to positive stimulation accompanying the initiation of movement. Neurons belonging to the second group begin to respond directly to acoustic stimulation after extinction of the conditioned reflex. Neurons of the mesencephalic reticular formation can thus exercise additional tonic ascending effects both in the production and inner inhibition of the conditioned reflex. The group of neurons with a phasic reaction, i.e., a double response (a direct response to sound and another produced by movement) displayed a drop in spontaneous activity during the shaping of inhibition of differentiation and of extinction in particular. It was found that the initial changes in the spike response of reticular formation neurons during conditioning and pseudo-conditioning are similar. There are thus grounds for stating that neurons of the mesencephalic reticular formation participate in the shaping, production, and inner inhibition of traditional and operant conditioned reflexes in a differentiated capacity rather than as a population reacting identically.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 161–171, March–April, 1986.     

Correlation between bioelectrical processes of the cortex, thalamus, midbrain reticular formation during formation of a defensive conditioned reflex in rabbits]

EEG power spectra of the sensorimotor area of the neocortex, the dorsal hippocampus, midbrain reticular formation and anteroventral thalamic nucleus, as well as corresponding coherence functions and phase spectra, undergo changes during formation and performance of defensive conditioned reflex in rabbit. The conclusion is draen that in the process of conditioning a morphofunctional system of brain structures is established including the above mentioned formations. Their functional integration occurs on the basis of theta-rhythm. The execution of a conditioned act requires isorhythmicity of electrical processes within the theta-range in the studied structures and an adequate level of their excitability, which is manifested in the dominance of 6,0 c/s frequency.     

Reproduction of electrographic correlates of a conditioned reflex after changes in the activity of the noradrenergic and dopaminergic systems

On the model of shortly delayed defensive conditioned reflex in cats, it was shown in acute experiments that pharmacologically elicited change of NA system activity (clonidine, 0.2 and 1.5 mg/kg intravenously) and of DA system activity (apomorphine, 3 mg/kg intravenously) leads to a definite manifestation of electrographic correlates of memory trace, i.e. of conditioned evoked potential (EP) in examined brain structures, as well as of conditioned neurographic response (CNR) and conditioned skin-galvanic reaction (SGR). The increase of the NA system activity causes a rise of the number of conditioned EPs in the reticular formation, hippocampus and preoptic area along with an enhancement of CNR reproduction. The increase of the DA system activity contributes to the appearance of conditioned EPs in the hippocampus, amygdalar complex and central gray matter, together with an enhancement of the reproduction of conditioned SGR.     

Neuron activities of the motor cortex during conditioned eye blink reflex in rabbits (author's transl)]

Effects of the tone (CS) on neurons of the motor cortex were investigated in naive, pseudoconditioned, and conditioned rabbits. Conditioning to eye blink reflex was made by a combination of CS and air puff (US). Effects of electrical stimulation of the subcortical structures were also observed on the cortical neurons associated with the conditioned reflex. The results were as follows. (1) Proportion of neurons which significantly increased the firing rate in response to the CS, type E, was higher in the conditioned group than in other two groups. On the other hand, no group difference was found in the proportion of neurons which significantly decreased the firing rate to the stimulus, type I. (2) Most of the type E neurons in the conditioned rabbits began to fire at latencies of about 50 to 100 msec after the CS, preceding about 200 msec to the appearance of the peripheral conditioned responses (EMG). (3) Most of the type E neurons in the conditioned animals were more easily affected by stimulation of the medial geniculate body and the brain stem reticular formation. Based on the results mentioned above, it is concluded that in the rabbits conditioned to the eye blink reflex, excitability of neurons in the motor cortex is enhanced by the tone (CS), and by electrical stimulation to the medial geniculate body and the brain stem reticular formation.     

Effect of diazepam on the impulse activity of neurons in the sensomotor and visual areas of the rat cerebral cortex

Acute experiments on nonanesthetized curare-treated rats with the recording of the neuronal activity of the cortex were conducted; a determination was made of the threshold doses in which diazepam influenced the spontaneous and induced activity of the neurons of the sensory-motor and optic cortex. Diazepam proved to depress the spontaneous and induced activity of the neurons of the sensory-motor and optic cortex. Diazepam proved to depress the spontaneous and induced activity of the neurons of the sensory-motor cortex in considerably lesser doses than the neuronal activity of the optic cortex. It is supposed that the neurons of the anterior portions of the cortex were more sensitive to diazepam than the neurons of the limbic structures and the reticular formation.     

Effect of GABA on a temporary connection in a neuronal population of the sensorimotor cortex in rabbits

Evoked activity of sensorimotor cortical neurones in response to stimulation of the pyramidal tract, medial lemniscus and reticular nucleus of the midbrain tegmentum; driving reaction of cortical neurones at stimulation of these brain structures of growing frequency, and conditioned reflexes elaborated by combination of direct stimulation of the sensorimotor cortex and electrocutaneous stimulation were studied in awake nonimmobilized rabbits. Application to the cortex of GABA solutions of low concentration (less than or equal to 1%) emphasizes the evoked neuronal responses, facilitates the appearance of driving reaction and contributes to the manifestation of the temporary connection. Application of GABA solutions of higher concentration (greater than 2%) leads to opposite effects. Positive correlation is found between electrical and behavioural phenomena. The described experimental approach may be used for analysis of various types of influences on temporary connection formation.     

Effects of transections and electrical coagulations in the medulla oblongata upon the activities in the respiratory muscles of the crucian carp (author's transl)]

The following conclusions may be drawn from the results in this work. The respiratory cycles are formed by the neuronal machinery in the reticular formation under the posterior part of the vagal motor nucleus. The motor neurones or the neuronal networks composing the motor nucleus of the respiratory muscles tonically discharge the action potentials, when the neurones or the networks are released from the inhibitory influences of the interneurones connecting the neuronal machinery to the motor neurones. Furthermore, the interneurones probably generate the tonic discharges after removing the inhibitory influences of the other interneurones or the neuronal machinery on them. A reflex mouth closing is elicited by a mechanical stimulus applying on the upper lip. The motor neurones of the m. adductor mandibulae are activated via only one synapse in the reflex. The reflex action potentials recorded from the motor nerve reduce in amplitude at the resting phase of the nerve in the respiratory cycles. These results suggest that the respiratory motor neurones are by nature spontaneous generators of the tonic action potentials and, in the time of the normal breathing, the tonic activity is interrupted by an inhibitory influence of the neuronal machinery generating the respiratory cycles.     

Response of recticular and dorsal thalamic nuclei neurons during extinction of conditioned implementation in the cat

Unit activity in 66 neurons of the reticular (R) nucleus and 31 neurons of the ventropostrolateral nuclei of the thalamus, and 14 neurons of the posterolateral nuclear complex, the pulvinar, were studied during extinction of the conditioned food implementation reflex. The number of R neurons that had responded to initial excitation in the first 300 msec after the conditional stimulus (CS) decreased with the extinction. Simultaneous disappearance of conditioned-reflex placement movements and late excitatory and inhibitory responses of R and dorsal thalamic nuclei neurons with latent periods exceeding 300 msec was also observed. Extinction of the conditioned reflex (CR) led to a significant lowering of background activity in two-thirds of investigated R and other thalamic nuclear neurons. This suggests that efferent effects from the reticular nucleus are decreased during Cr extinction.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the USSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 1, pp. 3–8, January–February, 1991.     

Generalization of an instrumental defensive conditioned reflex elaborated to electric stimulation of the hypothalamus in dogs

Generalization of defensive conditioned reflexes elaborated to electrical stimulation of the lateral hypothalamus (LH) was studied in four dogs with electrodes implanted in various structures of the limbic system. Electrocutaneous stimulation was switched off when the dog lifted the foreleg to a definite level. Generalization of the conditioned reflex was manifested in different degrees when testing different formations of the limbic system, or testing one and the same structure, but at different stages of conditioning. Two types of generalization were found: the first one--a well pronounced motor reaction, by its latency, level and duration of lifting the foreleg similar to movements appearing in response to the conditioned stimulus; and the second one--low amplitude or short-term movements differing from conditioned ones. The first type of generalization was observed in response to stimulation of LH, contralateral to the point of signal stimulation, of the ventromedial hypothalamic nucleus, the mammillary bodies and the basal, lateral and to a lesser degree, the central nucleus of the amygdaloid complex; the second type--in response to stimulation of the ventral hippocampus, the medial and lateral septum nuclei.     

Postnatal development of the scratch reflex in rabbits]

The development of the scratch reflex was studied in newborn (up to 2 months old) rabbits in norm and after elimination or activation of some parts of their nervous system (reticular formation, cerebellum, caudate nucleus, cerebral cortex, superior cervical sympathetic ganglia). The experiments with the section of the brain stem at the border between the medulla and the midbrain showed that in very young (5-10 days old) rabbits in norm the scratch reflex is controlled by the spinal cord with no influences of structures situated above the section's level. Later on the spinal mechanism of the scratch reflex becomes subject to supraspinal influences, among which in 2-3 weeks old animals facilitatory effects are predominant produced, in particular, by the reticular formation and the cerebellum, whereas in older age prevail inhibitory influences of the cerebral cortex, cerebellum, caudate nucleus and the sympathetic nervous system.     

Alterations of spectral parameters of rat brain electrical activity after sciatic nerve section

Changes in electrophysiological brain characteristics accompanying the development of neurogenic pain syndrome induced by transsection of sciatic nerve were analyzed. At the maximum pain syndrome 3 weeks after the deafferentation, a reorganization of the brain electrical activity was observed in the limbic structures (hippocampus, amygdale, and nucleus accumbens), frontal cortex, and the caudate putamen. An increase in the relative spectral power of the delta and alpha bands and a decrease in the relative power of the beta2 band (as compared to baseline activity) took place. Alteration of the electrical activity in the limbic structures did not depend on manifestations of the neurogenic pain syndrome (autotomy). The increase in the relative spectral power of the alpha-band activity in all the structures under study suggests the involvement of the reticular thalamic nucleus in pathogenesis of neurogenic pain syndrome.     

Intralimbic evoked potentials in the formation of an instrumental food conditioned reflex to hippocampal electrostimulation in dogs]

In dogs with the electrodes implanted in the hippocampus, amygdala, septum and hypothalamus an instrumental alimentary conditioned reflex (CR) was elaborated to electrostimulation of the hippocampus. Intralimbic evoked potentials (EPs) were studied during the elaboration and extinction of this reflex and during stimulations of limbic structures conducted with the purpose of CR generalization checking. Late EP components in the lateral hypothalamus and central nucleus of the amygdala changed during CR elaboration and extinction and in the hippocampus during amygdala testing. In both cases the amplitude of trace positivity and of slow negative wave was less, when during stimulation of the structure an instrumental movement was initiated than at its absence.     

Distribution of 1,25-dihydroxyvitamin D3 receptor immunoreactivity in the limbic system of the rat.

We used immunocytochemistry to obtain a complete cellular and subcellular mapping of the 1,25-dihydroxyvitamin D3 receptor protein (VDR) in the rat limbic system. We observed specific VDR immunostaining in the nucleus as well as in the perinuclear cytoplasm of neuronal cells. The limbic system consists of a variety of neuronal structures, and is known to have influence on memory, behavior, emotions and reproduction. In the hippocampal formation, we found strong nuclear staining as well as less distinguished cytoplasmic VDR staining in CA1, CA3 and CA4. The CA2 area showed a unique cytoplasmic predominance of VDR. The amygdala was found to exhibit specific patterns of VDR distribution in the various regions of the nucleus. We observed distinct differences of VDR localization within the limbic preoptic areas of the hypothalamus. Further parts of the brain we analyzed included the mammillary bodies, the indusium griseum and the cingulate cortex. The subcellular distribution of VDR in regions of the limbic system suggests a specific functional role of the receptor protein and indicates a role for calcitriol as a neuroactive steroid.     

The correlations of the evoked activity of the auditory cortex and amygdala in cats during conditioned reflex activities]

By averaged summary activity of symmetric points of the cat auditory cortex and amygdala evoked by sound, the character of interaction (Spirman coefficient of correlation) was determined between the structures before, at elaboration, at extinction and restoration of instrumental food-procuring reflex and also at automatization of the reflex. Individual character of co-tuning of the cortex and amygdala was found at adaptation of the animal, changing into close interconnections at formation of the reflex and into disturbance of their correlation at its extinction. Stabilization of the reflex up to the stage of automatization was accompanied by the weakening of connections between the cortex and amygdala. The results testify to complex and dynamic character of interaction in the system cortex-amygdala in conditioned activity.     

Site on inhibition of the spino-bulbo-spinal reflex on stimulation of the anterior lobe of the cerebellum.

The effect of stimulation of the anterior lobe (AL) of the cerebellum on spinobulbo-spinal (SBS) reflex activity evoked by somatic and splanchnic afferentation was studied in chloralose-anaesthetized cats. Unit activity synchronous with the SBS component of the efferent discharge was observed at the level of motoneurons, descending axons of the dorsolateral and ventral funiculi and neurones of the reticular formation (RF). Conditioning stimulation of the AL inhibited this unit activity. Reticular formation units influenced from the AL had direct contact with segmental structures; these results showed that disappearance of the SBS reflex during AL conditioning is associated with the depressant effect of the cerebellar cortex on the reticular formation.     

Brainstem areas involved in the aspiration reflex: c-Fos study in anesthetized cats

Expression of the immediate-early gene c-fos, a marker of neuronal activation was employed in adult anesthetized non-decerebrate cats, in order to localize the brainstem neuronal populations functionally related to sniff-like (gasp-like) aspiration reflex (AR). Tissues were immunoprocessed using an antibody raised against amino acids of Fos and the avidin-biotin peroxidase complex method. The level of Fos-like immunoreactivity (FLI) was identified and counted in particular brainstem sections under light microscopy using PC software evaluations in control, unstimulated cats and in cats where the AR was elicited by repeated mechanical stimulation of the nasopharyngeal region. Fourteen brainstem regions with FLI labeling, including thirty-seven nuclei were compared for the number of labeled cells. Compared to the control, a significantly enhanced FLI was determined bilaterally in animals with the AR, at various medullary levels. The areas included the nuclei of the solitary tract (especially the dorsal, interstitial and ventrolateral subnuclei), the ventromedial part of the parvocellular tegmental field (FTL -- lateral nuclei of reticular formation), the lateral reticular nucleus, the ambigual and para-ambigual regions, and the retrofacial nucleus. FLI was also observed in the gigantocellular tegmental field (FTG -- medial nuclei of reticular formation), the spinal trigeminal nucleus, in the medullar raphe nuclei (ncl. raphealis magnus and parvus), and in the medial and lateral vestibular nuclei. Within the pons, a significant FLI was observed bilaterally in the parabrachial nucleus (especially in its lateral subnucleus), the Kolliker-Fuse nucleus, the nucleus coeruleus, within the medial region of brachium conjunctivum, in the ventrolateral part of the pontine FTG and the FTL. Within the mesencephalon a significantly enhanced FLI was found at the central tegmental field (area ventralis tegmenti Tsai), bilaterally. Positive FLI found in columns extending from the caudal medulla oblongata, through the pons up to the mid-mesencephalon suggests that the aspiration reflex is thus co-ordinated by a long loop of medullary-pontine-mesencephalic control circuit rather than by a unique "center".     

Cytoarchitecture of the formatio reticularis of the brain stem of the dolphin]

In the present study some qualitative and quantitative features of the reticular formation of the medulla oblongata, pons and midbrain have been elucidated by cytoarchitectonic methods in the dolphin (Tursiops truncatus). The studies have demonstrated that similar to land mammalia, the dolphin has a reticular formation made up of spatially open cell groups lying in the deepest parts of the brain stem. Cytoarchitectonically the component parts of the reticular formation show a number of peculiarities enabling us to distinguish separate nuclei. In the dolphin peculiar architectonics have been observed in the nucleus gigantocellularis medullae oblongatae, nucleus papillioformis or the nucleus reticularis tegmenti Bechterewi and the nucleus centralis superior medialis seu ventralis. Fairly poor in cells are the nucleus centralis caudalis pontis and the nucleus centralis oralis pontis. We failed to single out as autonomous nuclei cell groups corresponding to the nucleus funiculi lateralis and the nucleus paratrochlearis of the land mammalia. The size and density of cells in nuclei have a number of peculiarities. The analysis of the ratios of the brainstem volume to that of reticular structures has shown them to be the smallest in the dolphin as compared with land mammals. The smaller share held by the brain-stem reticular formation and its cytoarchitectonic features can be associated with the functional properties resulting from the greater specialization of some of brain-stem systems (e.g. auditory, vestibular, extrapyramidal etc.) in the dolphin in comparison with land mammals.     

Immunohistochemical demonstration of c-fos protein in neurons of the medulla oblongata of the musk shrew (Suncus murinus) after veratrine administration.

We subcutaneously injected 0.5 mg/kg veratrine into the musk shrew (Suncus murinus), observed the presence or absence, latency, and the incidence of vomiting in each animal for 90 min, and selected animals that frequently vomited (FV group) and those that did not vomit (NV group). Subsequently, animal brains were removed, and the induction of c-fos protein (Fos) was immunohistochemically examined to evaluate neuronal activity in the medulla oblongata. The distribution of Fos-positive neurons in the medulla oblongata was similar between FV and NV groups, with numerous neurons along the entire length of the nucleus of the solitary tract and in the ventrolateral reticular formation. Both veratrine-injected groups showed higher numbers of positive neurons than the saline administered group. However, while the FV group showed a high concentration of positive neurons in the dorsal-dorsomedial reticular formation of the nucleus ambiguus in the rostral medulla, the NV group showed few positive neurons in this area. Fos activity in neurons in this area appeared to be higher in animals with a higher incidence of vomiting.