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Brain Research Bulletin, Vol. 37, No. 4. pp. 377-389, 1995 Copyright Q 1995 Elsevier Science Ltd Printed in the USA. All tights reserved

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Neuronal Regeneration and Estrous Cycle Restoration After Locus Coeruleus-periventricular Gray

Substance Section

LUIS PASTOR SOLANO,FLORES,*’ MARTHA PATRICIA ROSAS-AFELl+NO,* ROSALINDA GUEVARA-GUZMAN,* LEON CINTRA-MCGLONEt AND SOFIA DIAZ-CINTRAt

*Departamen to de Fisiologia, Facultad de Medicina, hive&dad National Autdnoma de M6xico. Ap. Postal 70250, 04510- Mhxico, D. F.

tlnstituto de lnvestigaciones BiomfSdicas, hive&dad National Autdnoma de Mbxico. Ap. Postal 70228, 045 1 0-Mbxico, D. F.

[Received 2 November 1992; Accepted 22 December 19941

ABSTFMCT: The locus coeruleus (LC) was anatomically sepa- rated from the periventrlcular gray substance (PVG) by means of kniie cuts in the adult female rat presenting regular estrous cycling. This resulted in a transient suppression of the estrous cycling that lasted lo-13 days after surgery. After thii period, irregular or regular cycling activity was observed. The regular cycling was restored 3045 days after the knife cuts. Golgi im- pregnation of some of the brains of these rats revealed regen- erative elements in the knife-cut-insulted area. Thus, blood ves- sels, macrophagic-like elements, and glial-like elements were observed in close relation with the knife-cut pathway. Addition- ally, well-defined stained neurons typical of the LC and PVG were observed In close proximity to the knife-cut pathway. Den- dritic and axon projections towards the insulted area were ob- served. Well defined axons were seen across the knlfe-cut path- way. These data support, first, that the LC-PVG communication is part of a circuitry for the modulation of gonadotropic actlvlty, and second, that in the restoratlon of the estrous cycliclty after the knlfe cut, regenerative processes leading to a LC-PVG func- tional reconnection occurred after the knii cut

KEY WORDS: Locus coeruleus, Perfventrfcular gray, Estrous cy- cle, Gonadotropic activity, Golgi, Neural plasticity, Neural tissue damage, Neural circuitry, Function restoration, Function impalr- ment, Noradrenergic nuclei, Rat

INTRODUCTION

The noradrenergic nucleus locus coeruleus (LC) provides ter- minals to hypothalamic structures involved in the neuroendocrine mechanisms for gonadotropic hormones release [25,28,29,37]. The LC noradtenergic influence on these mechanisms has been widely studied, and the phenomenon seems to be very complex. For instance, an inhibitory role of noradrenergic synapses in the modulation of luteinizing hormone (LH) release has been sug- gested, because the electrical stimulation of the ascending nor- adrenergic bundle-which includes the LC ascending axons

[60]-either completely or partially inhibits the characteristic pulsatile pattern of blood LH levels in the ovariectomized rat [31]. Additionally, electrochemical stimulation of the LC in freely behaving rats on the day of proestrous blocked ovulation and the spontaneous surge of LH. Also, in ovariectomized, es- trogen-primed rats, the LC electrochemical stimulation blocked the release of LH triggered by electrical stimulation of the medial preoptic area (MPOA) [ 161. In contrast to these, the existence of a stimulatory noradrenergic projection from the LC to the MPGA has been recently reported that seems to operate under particular conditions of excitability of the MPGA because, following LC electrical stimulation, the LH surges induced by MPGA electro- chemical stimulation were significantly augmented and main- tained above those levels obtained after MPOA electrochemical stimulation alone, whereas LC stimulation alone was totally in- effective in altering basal LH concentrations [ 191.

The complex participation of the LC terminals upon the hy- pothalamic mechanisms for gonadotropic release illustrated by the above-mentioned facts, consequently, suggests an involve- ment of equally complex neural circuities. In this regard, exper- iments performed in our laboratory directed to explore the influ- ence of LC upon the circadian activity-related structures [i.e., the suprachiasmatic nucleus (SCN)] participating in the expression of the gonadotropic activity, provided evidence supporting that another structure-the periventricular gray substance (PVG) ad- jacent to the LC-might also be involved in the gonadotropic mechanisms. Thus, the persistent estrous induced either by ex- posure to constant bright light [54] or induced by damage of the SCN [SS] is transiently suppressed after the electrolytic damage of the LC. Also, the electrolytic damage of LC results in a tran- sient cessation of the rhythmic estrous cyclicity in the sexually naive rat [53.54]. In these studies it was also shown that estrous cyclicity-or persistent estrous-suppression effects after LC electrolytic damage-was prevented by simultaneous electro- lytic damage of the PVG adjacent to the LC. On the other hand, anatomical relationships between the LC and the PVG have been

’ Requests for reprints should be addressed to Dr. Luis Pastor Solano-Plores, Department of Physiology, Health Sciences Centre, The University of Western Ontario, London, Ontario, Canada N6A X1.

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378 SOLANO-FLORES ET AL.

A

LOCUS CGERULEUS-PERIVENTRICULAR GRAY

described. Thus, a diffuse interstitial plexus that lies in the ven- trolateral angle of PVG adjacent to the LC has long been known to send afferent connections to the LC [44]. Also, LC dendritic branches extend into the PVG [11,22,33,50,57]. Recently, LC axons have been observed in the PVG [ 181.

With the above in mind, it is worthwhile to think that a LC- PVG functional interaction might be implicated in the control mechanisms for gonadotropic activity. Should this interaction ex- ist, then, any impairment of the LC-PVG communication should be reflected in the gonadotropic activity. To test this, the present study was focused to determine the effects of anatomical sepa- ration of the LC from the PVG upon the estrous cyclicity in the female rat by means of knife cuts.

METHOD

The experiments were performed using Wistar female rats. The animals were mature virgins weighing from 200 to 300 g at the beginning of the experiment. The rats were housed in indi- vidual cages. The temperature was kept at 22 + 2°C. The rats were maintained under a 12 h lights on/lights off schedule. An- imals had free access to food and tap water. To obtain vaginal smears, vaginal lavages were performed daily from 1400 to 1600 h using 0.15 ml of 0.9% NaCl solution with a modified Pasteur pipette 1 mm o.d., 3 mm length of tip bent perpendicularly to the main shaft to introduce just the tip and to avoid damage or ov- erstimulation [53,54]. The dried vaginal smear samples were stained with a Grtlndwalds-Giemsas Azur Eosin Methylenblau mixture (Merck) in methanol, rinsed in phosphate buffer pH = 7.4, rinsed with tap water, and coverslipped. Vaginal smears were examined with the aid of a Polyvar (Reichert Jung) microscope under bright-field illumination at 400X magnification. The sam- ples were labeled according to any of the four estrous cycle phases (proestrous, estrous, metaestrous, or diestrous) or inter- mediate stages [53]. These data were recorded on estrous cyclic- ity charts. The ordinate of each chart represents the eight stages for all of the successive days of the experiments. Each sample was represented with a point in the chart. Six or more cycles were recorded as a control before the LC was anatomically sep- arated from the PVG, by means of a stereotaxic knife cut. Sur- gical procedures were performed under chloral hydrate anesthe- sia (400 mg per kg, IP). Xilocaine paste was applied as local anesthetic to the tissue points contacting the stereotaxic frame. A 1 mm width, 250 pm thick double-edge iron blade was used to perform the knife cuts. The blade was manufactured by grind- ing a hacksaw piece (2 cm length) with an emery grinder. The cutting edges and the point were razor-blade sharpened. The pen- etrating end of the blade was sharp-point shaped. The blade was set vertically on a stereotaxic holder and the two cutting edges were oriented following the anterior posterior direction of the stereotaxic frame. The tip of the blade was directed towards the following coordinates: P = 8.6-8.8 from bregma in the horizon- tal skull, L = 0.67, and H = 6.5-7.0 from the brain surface. From that point, the blade was sliced 1 mm forwards and 1 mm backwards following the anterior-posterior direction. Knife cuts

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were made bilaterally. After the blade was withdrawn, a small piece of surgical gelatine foam was put in the hole of the skull, and the skin wound closed with cotton surgical suture stitches. Five sham operated rats served as controls. In the sham knife-cut rats all the surgical procedures were made, but the tip of the blade was left 3.0 mm above the target. Surgical procedures were car- ried out antiseptically. No antibiotic treatment was applied after surgery. At the end of the experiment, an overdose of chloral hydrate was applied, and the rats were intracardially perfused with isotonic saline solution followed by 10% formalin. Brain sections, 50 pm of thickness were obtained and stained with cre- syl violet. The location of the knife cut was determined with the aid of a stereotaxic atlas [40]. Some brains were prepared for a classical rapid Golgi impregnation as described elsewhere [15]. Camera lucida drawings were made, and color photographs were taken from selected sections (Kodacolor Gold 100). Animals that presented weakness, respiratory or gastric diseases or motor im- pairment after surgery were discarded.

RESULTS

The results are based on 23 rats that survived at least 15 days postsurgery and that maintained a healthy aspect. Approximately 60% of the animals died after surgery. Regular estrous cycles of 4-5 days could be recorded in the rats prior to surgery. The LC- PVG knife cut resulted in a suppression of the estrous cycling activity (Fig. 1). In general, the effect began as soon as the pre- knife-cut cycle was over. The estrous cycling suppression en- dured IO- 13 days. During this period, the vaginal smear was typically diestrous. Then, the estrous cycling activity was rein- stalled. In some rats, this cycling reinstallation was characterized by the presence of three to six irregular cycles before regular cycling was reestablished (Fig. 1 A, rat 5). Some other rats pre- sented regular cycling immediately after the suppression period (Fig. 1 A, rat 14). Some rats presented an additional period of cycling suppression before regular cycling was reestablished (Fig. 1 A, rat 87). In general, regular cycling was reestablished 30-45 days after the knife cuts. Only two out of 25 rats in which a successful LC-PVG knife cut was performed did not present the estrous cycling suppression effect. Sham-operated animals kept cycling regularly throughout of the experiments. The his- tological analysis of the brains of all the rats that presented dis- turbances of the estrous cycling activity revealed a successful LC-PVG knife cut (Fig. 1B) that essentially involved the totality of the LC-PVG interface. In those few cases that some LC- PVG interface was spared by the knife cut, no apparent corre- lation could be found between the pattern of cycling reinstallation and the extension of the knife cut (Fig. lA, rat 14 and rat 87). Additionally, the knife-cut-insulted area presented abundant and well-defined neural elements along and within the knife pathway as revealed by cresyl violet staining (Fig. 1C). This fact suggested that some sort of healing phenomena in the insulted tissue had occurred alter the knife cut. This led us to prepare some brains of these rats for Golgi impregnation to obtain a more detailed picture of the knife-cut-insulted area.

FIG. 1. Effect of LC-PVG knife cuts upon the estmus cyclicity in the rat. (A) Bstrous cyclic@ charts from LC-PVG knife-cut rats. P: proestmus, E: est~~us, M: metacstrous and D: diestmus. Intermediate stages are also indicated. Each black dot represents the corresponding stage of the vaginal lavage for that day. The E stage is indicated by a continuous horizontal line. Lower left labels are the rat identification numbers. ‘Ilie black vertical arrow at the bottom of each chart signals the day at which the LC-PVG knife cut was made. (B) Series of histological drawings of coronal sections taken through the region of the J_C of the rats whose estrous cyclicity charts are shown in A. llre drawings extend rostmcaudally from btegma -9.16 (left) to -10.3 (right) [40]. The vertical lines represent the pathways followed by the knife to reach the dorsal brain stem and separate the LC-PVG junction. Lower left labels are the rat identification numbers. (C) Bright-field photomicrog-raph of a cre~yl violet-stained comnal hemisection at LC level (bregma -9.68 to -9.8 [40]). Rat 5. Vertical downwards arrow signals the pathway of the knife cut separating LC and PVG. Curved arrow signals the ventral end of the knife cut. Note the dense accumulation of somata along and within the knife cut. An amplification of this area is presented in the inset. LC: locus coeruleus. PVG: periventricular gray substance. MS: mesencephalic trigeminal nucleus. 4~: fourth ventricle. Calibrations: 500 pm, and 100 pm for the inset.

PIG. 2. Axon from a PVG neuron crossing the LC-PVG knife cut-regenerated area. Rapid Golgi stain. Bat 14. (A) Photomontage depicting a panoramic view of a mesencephalic coronal hemisection at LC level (bregma -9.3 to -9.68 [40]). The three vertical downwards arrows show the pathway followed by the knife through the inferior collicuhts (IC) across the fourth ventricle (4~) reaching the floor of the 4v at the LC-PVG junction to penetrate in the brain stem tissue (about 440 Mm). (B) Closeup of the area signaled by the lower downwards arrow at panel A. White backgrounded arrowheads enclose the knife-cut-regenerated atea between the LC and the PVG. The curved arrow signals a typical pyriform neuron from the PVG whose axon crosses the LC-PVG knife-cut-regenerated area. (C) Closeup from panel B. The white backgrounded arrowheads enclose the knife- cut-regenerated area. Note the lower optical density and the wood grain-like aspect of this area. Two blood vessels (v) run parallel at both sides of the lower part of this area. The vertical downwards arrow signals the axon from a PVG neuron at the site of crossing the LC-PVG knife-cut- regenerated area. The field encompassed by the white interrupted frame is presented in panels D and E at higher magnification and different focus. (D) white backgrounded arrowhead signals the axon from a PVG neuron before crossing the knife-cut-regenerated area. (E) White backgrounded arrowhead signals the same axon shown in panel D just in the site where the knife cut was made, following its course to the LC. LC: locus coeruleus. PVG: periventricular gray substance. LPB: lateral parabrachiaf nucleus. SCP: superior cere%eIIar peduncle. MS: mesencephaiic trigeminal nucleus. Calibration: A: 1000 km. B: 500 Wm. C: 100 Mm. D and E: 10 pm.

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FIG. 3. High magnification photomontage depicting a panommic view of the field encompassed by the two white back- grounded arrowheads shown in Pig. 2B. These arrowheads are also shown here. ‘Ihe black downwards anew in the upper part of the picture signals the site in the floor of the fotuth ventricle (4~) at which the. knife penteated the brain stem tissue to separate the PVG from the LC. Ihe vertical white backgrou~M downwards arrow signals an axon originated at a PVG piriform neuron (P). at the site of crossing the LC-PVG knife cut regenerated area on coume to the LC. Note the. abundant neuropile in the tissue surrounding the knife-cut-regenerated area. Two blood vessels (v) run parallel at both sides of the lower pact of this area. pp: another piriform neuron. d: dendrites. g: glial elements. Calibration: 50 pm.

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382 SOLANO-FLORES ET AL.

FIG. 4. LC-PVG knife-cut-regenerated area containing angiogenic-, gliogenic- and neuron-like elements that conformed the presumptive regeneration processes triggered after the knife cut. Rat 87. Rapid Golgi stain. (A) Panoramic view of a coronal section at the level of the largest extension of the LC-PVG knife cut (bregma -9.3 [40]). The two black downwards arrowheads signal the symmetrical sites on the floor of the fourth ventricle (4~) at which the knife penetrated the brain tissue. The white backgrounded asterisk is at the upper right of a blood vessel. This same vessel is signaled in panels B, C, and D, and in Fig. 5. (B) Photomontage at higher magnification of the brain tissue beneath the floor of the 4v shown in panel A. The two white backgrounded stars encompass the pathway followed by the knife separating the right LC from the PVG. Note that the pathways on both sides of the brain are characterized by high density of silver deposits and silver stained elements. White backgrounded arrowheads signal. respectively, a typical pyriform PVG neuron and a typical multipolar LC neuron, which are in close proximity to the knife-cut-regenerated area. Both neurons send their axons to this area (as shown in panel D and in Fig. 5). Note that branches from the vessel labeled with the asterisk penetrate or run parallel to the knife-cut-regenerated area. Curved arrow signals a LC fusiform neutvn (FN). (C) Photomontage at higher magnification of the area surrounding the vessel labeled with the asterisk in panel B depicting a LC fusiform neuron (FN) that projects a fiber (FF) to the knife-cut-regenerated area. The pathway followed by FF is signaled with the four small upwards black arrowheads from right to left. The one at the extreme left signals the site at which FF arrives to the knife-cut-regenerated area. The white backgrounded downwards arrowhead signals the pathway followed by the knife. Note the homogeneity of the background tissue in both, knife-cut-regenerated area and surrounding tissue. (D) Photomontage at higher magnification of the area encompassed by the stars in panel B showing details of the angiogenic-. gliogenic- and neuron-like elements in the knife-cut-regenerated area. White backgrounded arrowheads signals, respectively, the same PVG and LC neurons as shown in panel B. AG: axon from the typical pyriform PVG neuron projecting to the knife-cut-regenerated are.a. AL: axon from the typical multipolar LC neuron projecting to the knife-cut-regenerated area. DN: LC neuron projecting dendrites to the knife-cut-regenerated area. N: LC ovoid neuron. F: nonidentified origin fiber with one end in the LC region and with the other end within the knife-cut-regenerated area (see the complete pathway of F in the camera lucida drawing shown in Fig. 5). DV: dendrite running parallel to a blood vessel (as shown in Fig. 5). FF: the same fiber as shown in panel C. VB: blood vessel within the knife- cut-regenerated area. g: glial elements. M: macrophagic-like elements. SCP: superior cerebellar peduncle. M5: mesencephalic trigeminal nucleus. MLF: medial longitudinal fascicuius. DR: dorsal raphe. Calibrations: A: 1000 pm. B: 500 pm. C and D: 50 pm.

LOCUS COERULEUS-PERIVENTRICULAR GRAY 383

RG. 4. Continued

384 SOLANO-FLORES ET AL.

FIG. 5. Camera lucida drawing of the LC-PVG knife-cut-regenerated area encompassed by the two stars, as shown in Fig. 4B and D, that summarizes the angiogenic-, gliogenic- and neuron-like elements that conformed the pre- sumptive regeneration processes triggered in this area after the knife cut. Abbreviations as in Fig. 4D. V: blood vessels containing some erythrocytes. S: small LC neuron projecting its fiber to the knife-cut-regenerated area. Calibration: 100 hm.

The analysis of the Golgi-impregnated brains revealed well- defined regenerative elements in the knife-cut-insulted area; thus, this area presented angiogenic-, gliogenic-, and neuron-like elements (Figs. 2, 3, 4, 5, and 6). The knife-cut pathways could be recognized by the high density of silver deposits and by the high density of silver stained cytological elements (Fig. 4). The knife-cut pathway was also characterized by a lower optical den- sity, that is, the typical yellowish color of the Golgi staining was less intense than that of the noninsulted tissue (Fig. 3). Also, the background tissue in the knife-cut area was characterized by a wood grain-like aspect parallel to the knife-cut pathway (Fig. 2). Blood vessels and capillaries were observed in close relationship with the knife-cut pathway. Some blood vessels were observed across the knife-cut pathway, some other running parallel to or within the knife-cut pathway (Figs. 2, 3,4, and 5). Macrophagic- like silver-stained elements were associated all along the knife- cut pathway (Figs. 4 and 5). Glial-like elements were also present

in the knife-cut pathway (Figs. 3. 4, 5, and 6). Well-defined stained neurons were observed in close proximity to the knife- cut area (Figs. 4,5, and 6). The shape and size of these neurons were typical to those described in healthy animals (according to [I I] and Fig. 7). Dendritic and axon projections to the insulted area were observed (Figs. 4. 5, and 6). Well-defined axons were observed across the insulted area (Figs. 2 and 3). In the rostra1 and caudal borders of the knife cut, well-stained neurons, glial elements, and numerous fibers were observed in close relation- ship with or superposed to the insulted area (Fig. 6).

DISCUSSION

The present work provides evidence indicating that the LC- PVC communication might be part of a modulatory circuitry for the gonadotropic mechanism. Thus, the anatomical separation of the LC from PVG by means of knife cuts resulted in a transient disruption of estrous cyclicity.

LOCUS COERULEUS-PERIVENTRICULAR GRAY 385

FIG. 6. Rostra1 border of the knife-cut-regenerated area of the same brain shown in Fig. 4. (A) Panoramic view of the section (bregma -8.8 to -9.16 [40]). The black arrowbeads signal the most rostra\ sites cut by the knife. (B) Photomontage at higher magnification from panel A. The fields encompassed by the white frames at the left and at the right are presented at higher magnification in panels C and D, respectively. (C) The white backgrounded arrows signal a LC fusiform neuron (FN) whose main dendrite (d) is superposed over the black background of the rostra1 border of the knife-cut pathway. FN: another LC fusiform cell sending dendrites to the insulted tissue. Note the numerous fibers with nonidentified origin in close relation with the knife-cut area. (D) The white backgrounded arrow signals a fiber crossing the rostra1 border of the knife-cut pathway. FN: LC fusiform neuron. MN: LC multipolar neuron sending dendrites to the regenerating tissue. g: glial elements. MLF: medial longitudinal fasciculus. 4v: fourth ventricle. Calibrations: A: 1000 pm. B: 500 pm. C and D: 50 pm.

386 SOLANO-FLORES ET AL.

Ad( eating knife I ments impref

FIG. 7. Photomicrographs of normal LC-PVG area (bregma -9.16 to -9.3 [40]) of a control rat observed at three different focus. Rapid Golgi stain. (A) Note the profuse neural fiber netting interconnecting LC and PVG. (B) Typical pyriform neuron (P) from PVG whose fiber crosses the LC-PVG interface. 0: ovoid LC neuron. (C) Typical multipolar (M) and fusiform (F) LC neurons. v: blood vessel. g: glial element. MLF: medial longitudinal fasciculus. 4v: fourth ventricle. M5: mesencephalic trigeminal nucleus. SCP: superior cerebellar peduncle. DR: dorsal raphe. Calibration: 500 pm.

ditionally, the present work also provides evidence indi- that regenerative neural processes were induced after the cuts. Thus, angiogenic-, gliogenic-, and neuron-like ele- were observed in the knife-cut brains, which were Golgi gnated.

Joining these two facts, the present work provides evic supporting that the restoration of estrous cyclicity, after the sient period of its suppression associated to the LC-PVG cuts, might be due to the reestablishment of a functional PVG neural connection.

Jence tran- knife LC-

LOCUS COERULEUS-PERIVENTRICULAR GRAY 387

HYPOTHALAMIC MECHANISMS cfm

LC-PVG COMMUNICATION I -11

GONADOTROPIC ACTIVITY A ‘FINE TONE’ CIRCUITRY FOR GONADOTROPIC

ACTIVITY REGULATION ?

FIG. 8. Histological evidence supports the existence of the circuitry shown above for gonadotropic activity control. Thus, to assess whether the LC-PVG communication participates in the gonadotropic mecha- nisms, the LC-PVG communication was interrupted by means of knife

Several studies indicate that LC and PVG are related to a number of structures involved in gonadotropic neuroendocrine control. The circadian organization of the estrous cycle [23,35,38] depends on the integrity of the SCN [8,21,43,47,48]. Electrophysiological [32] and scarce anatomical [9,42,45] evi- dence supports the existence of a bidirectional communication between the LC and SCN area that includes the paraventricular nucleus of the hypothalamus [2,5,6,25,26,46,52,56,58,59,61,62] and MPOA [5,9,12,25,30,42,49,56] in a multisynaptic cir- cuitry. On the other hand, PVG involvement with gonado- tropic mechanisms is supported by histological evidence [5,61]. Finally, the mutual relationship of both, LC and PVG, with the hypothalamus, can be linked together with the bidirectional anatomical connection between LC and PVG [ 11,18,22,33,44,50,57] in a circuitry for the modulation of go- nadotropic activity, as outlined in Fig. 8 in a comprehensive form. Due to the complexity of this circuitry, the LC-PVG knife-cut-suppressing effect upon the estrous cyclicity might involve a multiple diaschisis phenomenon [34]. In conclusion, the estrous cyclicity suppression observed in the LC-PVG knife-cut rats can be explained in terms of an impairment of the circuitry proposed above.

Analysis of Golgi-stained brains of the LC-PVG knife-cut rats indicated that regenerative events in the neural tissue affected by the knife cut were triggered. The neural tissue in the insulted area and the tissue surrounding the knife-cut pathway presented a well-defined aspect after Golgi staining, and blood vessels, macrophage-like, and glial-like elements were associated to the insulted area. These facts indicate that after the knife cut, differ- ent remodeling processes were triggered off in such a way that the tissue environment was favorable enough to maintain the neu- ral cells in healthy conditions, as can be inferred by the presence of multiple well-defined neurons in close proximity to the knife- cut area. Because a separation of the wound margins was not observed in the knife-cut brains, and scar tissue was not present in the knife pathway, it should be concluded that those remod- eling processes resulted in a successful closure of the wound. A complete closure of a wound after a brain knife cut as the one used in the present study was reached within 7- 14 days after the knife cut [36]. The importance of restoration of the blood supply for normal function of neuronal tissue surrounding a site of injury and the response of macrophages to injury of the central nervous system has been reviewed [20,41]. Additionally, the microenvi- ronment of the perivascular connective tissue leads to axon re- generation, while sustained growth and establishment of func-

tional axon terminals depends on the presence of glial cells [ 141. The neurons in the knife-cut-regenerated area presented a well- defined Golgi staining, and their shapes and sizes were similar to those of typical neurons from the same area as can be observed in healthy animals ([l I] and Fig. 7). These facts indicate that the biochemical activity of these neurons after the knife cut was maintained healthy enough to respond to injury in such a way that they reestablished their dendritic and axonic projections, as can be inferred by the presence of axons and dendrites within the knife-cut pathway. It is likely that biochemical processes similar to those reported elsewhere [10,24,63] could have happened in the neurons affected by the LC-PVG knife cut. Although the failure of axon regeneration in the central nervous system after injury to mammalian brain has been discussed [4,5 l], impressive evidence indicates that neurons in the central nervous system are capable of extensive axonal growth when an appropriate envi- ronment is provided [ 1,7,13,39] and that, in the adult rat brain, readily spontaneous regeneration of cut axons occurs that even cross the cut [ 171. The fibers of nonidentified origin in the rostra1 and caudal borders of the LC-PVG knife cut suggests that these fibers might well be detour fibers as those observed elsewhere after brain knife cuts [ 171. In conclusion, it is then conceivable that regenerative processes leading to an LC-PVG reconnection occurred after the knife cut.

Following IO- 13 days of estrous suppression effect by the LC-PVG knife cut, estrous cyclicity was reinstalled and, in gen- eral, irregular cycles were observed before the reestablishment of a regular pattern of estrous cyclicity. The regular cycling was restored 30-45 days after the knife cuts. Although vicarious functioning [34] of some other structures and/or supersensitivity phenomena [3,27] cannot be discarded as causes of the restora- tion of the estrous cyclicity, the regeneration elements observed in the Golgi-stained brains strongly suggest that an LC-PVG functional reconnection occurred after the knife cut, and so the restoration of the estrous cyclicity might then be partially ex- plained in terms of this reconnection. Although the present data do not allow us to directly establish the precise moment at which this reconnection was active, the temporal pattern displayed by the events of estrous cyclicity suppression and estrous cyclicity restoration deserves some comments. A complete wound closure is reached within 7- 14 days after a brain knife cut, and recovery of neurotransmission-related enzymatic activity distal to the tran- section is observed l-3 months after the operation [36]. Addi- tionally, 3-8 days after a brain tissue cut, detour fibers appeared that always seemed to reconnect the corresponding severed path- ways [ 171. Furthermore, at a brain wound site, marked angiogen- esis occurs approximately at day 5 after injury, and debris clear- ance from the wound site is almost complete at day 15 after the injury [20]. The period of estrous suppression in the LC-PVG knife-cut rats fits in well with these data, so it might be assumed that similar events could take place in these rats and, therefore, the period of estrous cyclicity suppression might reflect the lack of activity of the LC-PVG communication during which wound closure and tissue regenerative processes were in progress lead- ing to a LC-PVG reconnection. After this period, some more time-probably l-3 months (according to [36])-should have elapsed before complete functional activity of the LC-PVG communication had been reestablished and the neurotransmis- sion-related activity had been restored. Because this period also fits in well with the fact that the LC-PVG knife-cut rats pre- sented regular cycles 30-45 days after the knife cut, then, the moment at which regular estrous cycling was established might be assumed to be the moment at which complete functional ac- tivity of the LC-PVG communication was accomplished. Fi- nally, as a corollary, the irregular pattern of estrous cyclicity ob- served after its suppression period can be explained, assuming

388 SOLANO-PLORES ET AL.

that the moment at which irregular cycles were present corre- sponds to the moment at which the LC-PVG reconnection was starting to he. functional. Alternatively, it might he that at that same moment, vicarious functioning of some other structures and/or a supersensitivity phenomena were also beginning. In any or all cases, that irregularity of estrous cycling might he taken as a reflection of incomplete function of the LC-PVG communi- cation. Once the LC-PVG communication was fully active, the estrous cyclicity became regular. It might he. then, that the role of the LC-PVG communication is to confer a sort of harmony and synchrony to the activity of the different structures involved in the gonadotropic function. In conclusion, the functional mean- ing of the LC-PVG communication might he thought of as a modulatory-fine tone -circuitry for the gonadotropic control mechanisms.

ACKNOWLEDGEMENTS

The critical comments by Sim6n Brailowsky and the technical assis- tance of A. Aguilar, A. Galvan P. Dumn, and I. Perez-Montfort are gratefully acknowledged.

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