ACTA NEUROBIOL. EXP. 1981, 41: 553-563

Lecture delivered at the Symposium "Brain and behavior" held in Jablonna near Warsaw

May 1981

INTERANALYZER TWO-WAY NEURONAL CONNECTIONS DURING ALIMENTARY CONDITIONING IN CATS

G. Kh. MERZHANOVA

Laboratory of Conditioned Reflexes, Institute of Higher Nervous Activity and Neurophysiology, USSR Academy of Sciences

Butlerowa 5a, 117485 Moscow, USSR

Key words: Neuronal activity, sensorimotor cortex, visual cortex, alimentary conditioning, extinction, cats

Abstract. Milk conditioned reflexes to low frequency electrostimulat- ion of optic tracts was examined in four cats. Multineuronal activity from visual and sensorimotor cortical regions was recorded under con- ditions of chronic implantation. Intra- and interanalyzer interneuronal connections were statistically analyzed by constructing crosscorrelo- grams. The comparison of the results of interneuronal interaction in untrained and trained animals showed an increase of one-way and two- way connections both of intra- and interanalyzer types. Neuroni cha- racterized by intermediate amplitude spike, more than other neurons, changed their network properties and were enriched by incoming and outgolng connections in both visual and sensorimotor cortex of the trair:ed animals. An increase in the total number of interneuronal con- nections after acquisition was due to an increase of connections from the sensorimotor to the visual analyzer.

INTRODUCTION

Although studies on the discharge activity of separate neurons have contributed much to the cellular physiology of behavior, the elucidation

of the mechanism of temporary connections remain largely inadequate, requiring new ways to solve this problem. Recently, many researchers have attended to studies on the workings of the microsystems of tho , three and more cells as a functional unit in the systemic activity c ~ f thc entire brain. Such investigations are possible because of the wid.. use of computers, thereby permitting methods for the statistical evaluation of interneuronal connections. The most promising approach, from our point of view, is the method for studying the interaction of neurons, both of adjacent and remote areas, by evaluating the intervals of their interaction. S~ecifically, this approach uses cross-interval histograms of two or more pulse flows.

The method for statistically evaluating interneuronal connections may serve as a sensitive and an objective procedure for the investigat- ion of those problems of conditioned reflex activity which require a quantitative assessment of intercentral integrations. Such problems include the neuronal mechanisms of two-way conditioned connections. I'volving the concept of Pavlov (16) concerning forward and backward conditioned connections, Asratyan regarded this phenomenon as uni- versal for all types of conditioned reflexes, and he considered two-way connections as a common neurophysiological principle (1, 2). In the pres- ent work, the method of cross-correlational analysis of pulse flows was employed in studies of the interaction of neuronal groups in two cor- tics1 regions involved in the formation of conditioned reflexes.

METHOD

Experimental. The experiment used four alert cats of both sexes. The alimentary conditioned reflex (CR) to low frequency electrostimulat- ion of the optic pathways (GLB or OT) was established using a method previously described (12, 13). Multineuronal activity was registered by a set of nichrome electrodes implanted in the visual (field 18, the cor- tical projection of the conditioned stimulus, CS) and sensorimotor cor- tical regions field 4 (the cortical projection of chewers and lingual mus- cles). Simultaneous recordings of multineuronal activity from the two cortical regions and the mechanogram of licking movements were made on magnetic tape (Nihon Kohden tape-recorder).

Fragments' of neuronal activity background recordings for 30 s in trained and untrained cats from the cortical areas were used for statis- tical processing. In trained cats, the 30 s fragments of neuronal activity recordings were chosen, which preceeded the CS evocation of distinctly identified CRs.

Selection of spikes. The analysis of multineuronal activity involved the devising of the algorhythm and complex programs for selecting spikes, their sorting, and the constructing of auto- and crosscorrelo- grams. The selection of spikes was determined by their form and ampli- tude. The amplitude of the spikes of the same form was assessed accord- ing to especially preset levels, differing by 25-30°/o. Three series of im- pulses with different amplitudes were made up. Neurons with large, intermediate, and small spikes were arbitrarily designated as types of neurons, 1, 2 and 3 respectively (Fig. 1).

L Ims

Fig. 1. Neurograms of visual and sensorimotor cortical areas. Numbers indicate spikes selected in the statistical analysis. Calibration - 1 ms.

Construction of crosscorrelograms. Dependent relations of two pulse series were studied by using crosscorrelograms. The computerized ana- lysis makes it possible to construct crosscorrelograms with right and left branches, i.e., from neuron A to neuron B, and from neuron B to neuron A, simultaneously. Extremes of such histograms reveal the most frequently encountered intervals of the neuronal interaction of two pulse series. In our experiment, the duration of the right and left branches of the crosscorrelogram was 100 ms with 1 ms bin. The signifi- cant extreme of the crosscorrelational function was determined as the one for which deviation of all the points was 30 relative to the mean value.

Crosscorrelograms were analyzed according to their types - sym- metrical and asymmetrical. Symmetrical crosscorrelograms were regard- ed at those that had a similar pattern of interval distribution in the left and right branches and had a long duration of the dependence index. Asymmetrical crosscorrelograms had a d i f f e r a t parttern of interval dis- tribution and a short duration of the dependence index. According t3

available data asymmetrical peaks of crosscorrelograms point to the existence of functional connection between neurons (3, 5, 14, 19).

In describing the material, we used crosscorrelograms of asymmetri- cal type, the extremes of which were above the confidence level, and we viewed them as excitatory types of the dependent relations between neurons. Autocorrelograms were made for each neuron and were used for interpreting the crosscorrelograms. The data were obtained from the analysis of 279 crosscorrelograms.

RESULTS

The conditioned reflex (CR) in our experiment consisted of licking responses in cats during CS action. However, in the course of training (up to 300 and more trials) the conditioned licking response disappeared, and the presence of temporary connection was revealed in tests present- ations of the CS without reinforcement. The CR were observed in 90- 100°/o of such cases.

In the present experiment, the fragments of background recol.di~;gs in three states of the animals were measured specifically: in intact cats, in the same cats with stable CRs, and during extinction of CR. Alt:>- gether 61 neuronal pairs were examined to identify intranalyzer con- nections and 97 for interanalyzer connections. Some neuronal pairs showed the interrelations between neurons (two-way connections), others had one-way connections, and still others were independent.

The network properties of a separate neuron were determined by the number of connections incoming and outgoing from each neuron (5). This parameter of the network properties was investigated both within the scope of one analyzer (visual cortex, sensorimotor cortex), the intranalyzer connections, and between two analyzers (visual cortex and sensorimotor cortex), the interanalyzer connections.

The number of incoming and outgoing intra- and interanalyzer con- nections after conditioning was found to differ considerably. The common feature for the neurons of both analyzers appeared to be their acquisit- ion of incoming and outgoing connections. I t was especially characteristic of the type 2 neurons (Fig. 2).

In analyzing the activity of neurons of the visual and sensorimotor analyzers a certain difference can be seen. The visual cortex was cha- racterized by the increase of the outgoing interanalyzer and the incoming intranalyzer connections. Type 3 neurons of the sensorimotor cortex was characterized by the reorganization of intranalyzer connections (the predomination of incoming over outgoing) and the disappearance of in- teranalyzer connections, which were manifested before conditioning.

Fig. 2. Dynamic changes in the number of incoming and outgoing intra- and inter- analyzer connections for separate neurons of the visual and sensorimotor cortical areas before (A) and after acquisition of conditioned alimentary reflexes (B) and during the extinction (C). 279 cross-correlograms for 97 neuronal pairs in three cats were analyzed. Squares, visual; rhombs, sensorimotor cortex. Arrows inside and outside the rhombs and squares - intra- and interanalyzer connections, respectively. Numbers - selected neurons in conformity with the amplitude of

their spikes: 1. large; 2. intermedian; 3. small.

We conducted CR extinction by presenting the CS without reinfor- cement every other minute. Conditioned effectory responses were distinct in the first applications of CS. After 20 presentation of the CS without reinforcement, the effectory conditioned responses were extinguished. The failure of the CR to manifest itself in five successive presentations served as the criterion of extinction. The analysis of the network pro- perties of the neurons in the visual and sensorimotor regions showed the dynamics of changes in the number of incoming and outgoing con- nections of the inter- and intraanalyzer characters.

Figure 2 illustrates the reorganization of connections after extinc- tion. Thus, the type 2 neurons of the visual cortex preserved the acqui- red intraanalyzer connections and decreased interanalyzer connections, as compared to their number after conditioning. The type 1 neurons preserved only incoming intraanalyzer connections and 10s interanaly- zer connections. Type 3 neurons, while preserving intraanalyzer connec- tions, lost almost all interanalyzer ones. In the sensorimotor cortex, a substantial reorganization of connections could be seen for all the

three neurons. Types 1 and 2 neurons lost their acquired interanalyzer connections. Type 3 neurons remained without interanalyzer connec- tions, while the number of intraanalyzer connections decreased. Thus, CR extinction did not lead to the disappearance of all of the acqu i r4 connections, but to their reorganization.

On the basis of these data on the number of interanalyzer excitatory connections as well as the identification of the direction of these con- nections in intact, in trained animals and in the same cats after CR extinction, we constructed the schemes of dependent interneuronal re- lations (Fig. 3) for microsyst~ms of three neurons of the visual and sensorimotor cortical areas. '

Fig. 3. Patterns of interanalyzer interneuronal connections before (A) and alter conditioning (B), and during CR extinction (C). For identifying interanalyzer con- nections for A, B, and C, 26, 57 and 14 neuronal pairs, respectively, were analyzed.

Other indications as in Fig. 2.

In considering interanalyzer connections, a substantial intensifica- tion of the interaction between the neurons under investigation and the appearance of two-way neuronal interactions with the functioning CR could be seen (Fig. 3). Two-u-ay interactions were manifested in pairs

2 V - 2 SM; 3 V - 2 SM; 3 V - 1 SM; 1 V - 1 SM. After CR extinction the one-way functional connection from type 2 neurons of the sensorimotor cortex to neuron 2 of the visual cortex were maintai- ned, and there appeared a functional connection from type 2 neurons of the visual cortex to type 1 of the sensorimotor cortex. Thus, interana- lyzer connections had specific features after CR acquisition and extinc- tion.

The counting of the total number of interanalyzer connections re- vealed that the percentage of their manifestations more than doubled (45 - against 210/0) in the trained animals (Fig. 4). However, the per-

M-V

Fig. 4. The percent distribution of manifestations of interanalyzer connections for visual and sensorimotor cortical areas before (A) and after conditioning (B). The number of identified connections, nonhatched column; connections from visu?l to sensorimotor neurons, horizontal hatching; from sensorimotor to visual neurons, inclined hatching. On the ordinates, O/o manifestation of connections. Thr: number

of all possible connections between the neurons was taken as 100°/o.

centage of interrelations from the visual to sensorimotor cortex increa- sed only by 5O/0 (22 - against 17°/o). Hence, the total number of inter- ceuronal connections increased a t the expense of considerable increase of connections from the sensorimotor to the visual analyzer (by 19"In) (Fig. 4).

Data were obtained on time intervals of neuronal interactions of the visual and sensorimotor cortex. Functions for the distribution of the number of identified neuronal connections for interanalyzer neuro- nal pairs with different intervals neuronal iilteractions before and after conditioning are presented in Fig. 5. The relationship shows the in- crease of interneuronal connections in the trained animals with the pre- dominance of SM-V connections having temporary intervals in the range of 20 ms (dotted line). Similar connections in smaller numbers were observed from visual to sensorimotor neurons (solid line), while in untrained animals they were absent. This situation is characterized

Fig. 5. The distribution of the number of identified neuronal connections with different time intervals of neunonal interaction for interanalyzer neuronal pairs within the scope of the analysis epoch of 100 ms before (A) and after (B) con- ditioning. Solid line, interneuronal connections from visual to sensorimotor neurons;

dotted line, from sensorimotor to visual neurons.

by the coincidence of time relations of interneuronal connections in the range of 100 ms and the opposite direction of the curves in the range of temporary interval at 40-60 ms.

DISCUSSION

The analysis of the crosscorrelational interneuronal relations sho- wed that after formation of the conditioned alimentary reflexes in cats, qualitatively new neuronal interactions of the cortical projections of the conditioned (visual cortex) and the reinforcing (sensorimotor cor- tex) stimuli were established. The results of our expesimtmt showed that after conditioning, the total number of interanalyzer excitatory connections increased more than two times. It should be noted that this increase mainly occurred due to a rise of the connections from the neurons of the reinforcing (sensorimotor cortex) analyzer to the neu- rons of the signal (visual cortex) analyzer. From our point of view these connections are manifestations of backward conditioned connec- tions, which are activated after acquisition of CR's. The effect of the reinforcing stimulus on the structures of the signal stimulus by back- ward conditioned connections consists, according to Asratyan (2), both in the creation and maintenance of increased excitation of these s'cruc- tures for detection of the signal and formation of the specialized CR.

Asratyan proposed the assumption (1) that intrafocal connections are formed both in the cortical CS projection and the reinforcing pro-

jection, and that as a result of the combined action of the two stimuli, the interaction between these foci contributes to the process of the for- mation of "local conditioned state" in each. The functioning of twa-way conditicned connections results in mutual reinforcement. The data ob- tained in the present experiments defined the latter assumption by the fact that the functional connections between the neurons 3f the two foci, in the first place, are multicomponent ones. This finding was reflected in various interneuronal combinations of th$se connections as well as in different time delays characterizing these connections. Second, the reinforcing action from the cortical structures of the sen- sorimotor to the visual analyzer, i.e., by the backward conditioned con- nections, is relatively more pronounced than by the forward direction, from the visual to the sensorimotor structures. This observation was apparently explained by the fact that in our experiment the interana- lyzer connections characterized the animal's tonic conditioned state, in which the function of backward conditioned connections is probably more significant than in realization of the reflex to CS.

It can be assumed that interanalyzer connection patterns are orga- nized in different ways for these two cortical analyzers, which confirms their difference in function. The visual analyzer should receive the information of the conditioned signal and should be in a state of incres- sed excitation to detect this signal. The sensorimotor analyzer "forms" the effectory reaction of the animal.

According to previous data (5, 6, 10, l l ) , the correlative ampi~tude of the neuronal discharge in multineuronal activity is connected with the neuron size when recorded by a large diameter electrode. Taking into consideration also a column structure of the projection cortical regions (7, 18), it may be assumed that cells with a large spike amplitude in the visual cortex, receiving powerful incoming connections, transfer the signal to the next column of the same analyzer. The large neuron of the sensorimotor region mainly works for organization of its own three-neuronal pool. I t is important to note the work of the type 3 neurons of the visual and sensorimotor cortical areas. The type 3 neu- rons of the visual area acquired outgoing and incoming interanalyzer connections with the neurons of the sensorimotor cortical area, whereas the type 3 neurons of the lsensorimotor cortex did not have outcoming connections. Apparently, the function of these neurons is different dur- ing the formation of the pattern of interneuronal connections during conditioning. The functional importance of type 2 neurons in both cor- tical areas is of special interest and requires further special study.

Interneuronal interanalyzer connections during CR extinction had their own specific pattern. For instance, the connection from type 2

neurons of the sensorimotor cortex to type 2 neurons of the visual cortex remained functioning. This confirms once more the concept of Pavlov and other authors (4, 15) that extinction of a conditioned positive reflex leads not so much to its destruction as to the acquisition of a new CR with "negative" reinforcement.

It is difficult to imagine the organization of such a behavior as the CR without involving other structures, especially limbic system struc- tures connected with the animal's alimentary behavior. Therefore, i t can be assumed on the basis of our data that interactions of neurons of different analyzers with various time delays reflect different connec- tion links through cortico-subcortical and cortico-cortical interactions. It may also be assumed that interactions between visual and sensorimo- tor neurons with short intervals interactions (in the range of 10-20 ms) result from cortico-cortical interactions according to the data (8) for an isolated cortical strip. This assumption points to the fact that transfer of excitation from the visual cortex to the sensorimotor cortex takes 10 ms. The role of the reticular formation and certain thalamic nuclel in spatial synchronization of cortical biopotentials as well as in CR acquisition have been shown in a number of papers (8, 9, 17). Such influence can determine the changes in the periods of excitation for all of the links of neuronal cortical chains, which is probably one of the requirements for pulsation spreading in the cortex (9).

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